Reduction and control of pH and soluble CO2 for optimal nitrification for domestic, industrial and municipal wastewater

ABSTRACT

The invention includes a method for the optimization of the soluble CO 2  concentration in the aeration basin of an activated sludge system, which significantly improves the specific growth rate of the nitrifying bacteria. The result is a reduction in capital and energy costs for municipalities. The rate of nitrification is a product of the nitrifying bacteria biomass concentration and the specific growth rate of the bacteria. In the activated sludge system, the biomass concentration is maintained at high concentrations by reducing the wasting rate. The specific growth rate is a function of the ammonium concentration and the environmental conditions. Here, the inventors show that growth of nitrifying bacteria is inhibited when the soluble CO 2  concentration is elevated beyond certain parameters. Elevated soluble CO 2  concentration also reduces the pH, which also impacts the rate of growth of nitrifying bacteria.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Nonprovisional application Ser. No. 12/711,525, filed on Feb. 24, 2010, entitled “Reduction and Control of pH and Soluble CO₂ for Optimal Nitrification for Domestic, Industrial and Municipal Waste”, now U.S. Pat. No. 8,545,700, which claims priority to U.S. Provisional Application No. 61/154,811 filed Feb. 24, 2009, entitled “Method of Optimizing Nitrification in Full-Scale Activated Sludge Systems”, which are incorporated herein by reference

FIELD OF INVENTION

This invention relates to methods of treating wastewater.

BACKGROUND OF THE INVENTION

Nitrification is the first step for the removal of nitrogen from wastewater, where ammonium (NH₄ ⁺) is oxidized to nitrate (NO₃ ⁻) by aerobic, autotrophic, nitrifying bacteria. These bacteria are thought to have slow growth rates and are sensitive to pH and temperature swings, making nitrification difficult to maintain in activated sludge systems (Mobarry et al. 1996; Wagner et al. 1996). The slow growth rate and associated nitrification rate requires a lengthy solids retention time (SRT), as much as 20 days. Previous work has demonstrated that the growth of some autotrophic bacteria is carbon limited (Dagley and Hinshelwood 1938; Green et al. 2002; Denecke and Liebig 2003). Inorganic carbon was found to be a limiting factor in biological nutrient removal (BNR) systems due to the low partial pressure of carbon dioxide (pCO₂) of the atmospheric air introduced, and the loss of CO₂ by stripping (Wett and Rauch 2003). These factors were reported to limit the bulk concentration of CO₂ in wastewater and consequently affect nitrification.

The art lacks, however, an evaluation of the effect of elevated pCO₂ on the specific growth rate of nitrifying bacteria using activated sludge from three different types of BNR processes: extended-aeration, Modified Ludzack-Ettinger (MLE), and Bardenpho.

SUMMARY OF INVENTION

The rate of nitrification is a product of the nitrifying bacteria biomass concentration and the specific growth rate of the bacteria. In the activated sludge system, the biomass concentration is maintained at high concentrations by reducing the wasting rate. The specific growth rate is a function of the ammonium concentration and the environmental conditions. Traditionally, the specific growth rate has been improved by increasing the dissolved oxygen concentration through the use of higher aeration rates and fine bubble diffusers. The concentration of the soluble carbon dioxide (CO₂) has been overlooked as a controllable parameter of importance for the growth of these autotrophic bacteria. It has been assumed that the soluble CO₂ concentration in aeration basins is non-limiting. Here, the inventors show that growth of nitrifying bacteria is inhibited when the soluble CO₂ concentration is elevated beyond certain parameters. Elevated soluble CO₂ concentration also reduces the pH, which also impacts the rate of growth of nitrifying bacteria. The invention therefore includes a method for the optimization of the soluble CO₂ concentration in the aeration basin which significantly improves the specific growth rate of the nitrifying bacteria. The result is a reduction in capital and energy costs for municipalities.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:

FIG. 1: Effect of dissolved CO₂ concentrations on pH. Numbers indicate the dissolved CO₂ concentration of the aeration basin.

FIG. 2. Basic schematic of an illustrative WWTP utilizing the MLE Process. The WWTP treats 12 MGD (Q) and is comprised of 3 parallel anoxic tanks and 6 parallel aeration basins (FIG. 3). Each aeration basin is 60 feet wide, 180 feet long and 15 feet deep. Aeration of 1,000-1,500 SCFM in these basins provides adequate dissolved oxygen levels of 3 mg/L across the entire length. The average MLVSS is 2,450 mg/L.

FIG. 3. Illustrative WWTP utilizing the MLE Process.

FIG. 4. Improvement in the specific growth rate of nitrifying bacteria as a function of the percent of the internal recycle flow treated through an air stripping technology.

FIG. 5. Proposed retrofit of WWTP (FIG. 2) utilizing the MLE Process with air stripping technology to reduce soluble CO₂ of the internal recycle wastewater.

FIG. 6. Proposed retrofit of WWTP (FIG. 3) utilizing the MLE Process with CO₂ stripping technology to reduce soluble CO₂ of 50% of the internal recycle wastewater.

FIG. 7. Proposed retrofit of WWTP (FIG. 2) utilizing the MLE Process with CO₂ stripping technology to reduce soluble CO₂ of the wastewater entering the Nitrification train. The nitrate rich treated wastewater is internally recycled to the anoxic basin for denitrification.

FIG. 8. Proposed retrofit of WWTP (FIG. 3) utilizing the MLE Process with CO₂ stripping technology to reduce soluble CO₂ of 50% of the wastewater exiting the aeration basin #2.

FIGS. 9A-D. Plots of NO_(x) (NO₂ ⁻—N+NO₃—N; blue) and NO₃—N (green), which correspond to the growth of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB), respectively, for MLE#1 activated sludge and a range of dissolved CO₂ concentrations (5 mg/L) and pH (6.5, 7.0, 7.5, and 8.0).

FIGS. 10A-D. Plots of NO_(x) (NO₂ ⁻—N+NO₃—N; blue) and NO₃—N (green), which correspond to the growth of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB), respectively, for MLE#1 activated sludge and a range of dissolved CO₂ concentrations (10 mg/L) and pH (6.5, 7.0, 7.5, and 8.0).

FIGS. 11A-D. Plots of NO_(x) (NO₂ ⁻—N+NO₃—N; blue) and NO₃—N (green), which correspond to the growth of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB), respectively, for MLE#1 activated sludge and a range of dissolved CO₂ concentrations (15 mg/L) and pH (6.5, 7.0, 7.5, and 8.0).

FIG. 12. Ammonium oxidizing bacteria (AOB) and Nitrite oxidizing bacteria (NOB) μ_(max,obs) of MLE#1 activated sludge for range of dissolved CO₂ concentrations (5, 10, and 15 mg/L) and pH (6.5, 7.0, 7.5, and 8.0). The bars represent the 95% confidence interval.

FIG. 13. High pH ammonium oxidizing bacteria (AOB) μ_(max,obs) of MLE#1 activated sludge for range of dissolved CO₂ concentrations (5, 10, and 15 mg/L) and pH (6.5, 7.0, 7.5, and 8.0). The red columns are actual values with large 95% confidence interval.

FIG. 14. Low pH ammonium oxidizing bacteria (AOB) μ_(max,obs) of MLE#1 activated sludge for range of dissolved CO₂ concentrations (5, 10, and 15 mg/L) and pH (6.5, 7.0, 7.5, and 8.0). The red columns are actual values with large 95% confidence interval.

FIG. 15. High pH nitrite oxidizing bacteria (NOB) μ_(max,obs) of MLE#1 activated sludge for range of dissolved CO₂ concentrations (5, 10, and 15 mg/L) and pH (6.5, 7.0, 7.5, and 8.0). The red columns are actual values with large 95% confidence interval.

FIG. 16. Low pH nitrite oxidizing bacteria (NOB) μ_(max,obs) of MLE#1 activated sludge for range of dissolved CO₂ concentrations (5, 10, and 15 mg/L) and pH (6.5, 7.0, 7.5, and 8.0). The red columns are actual values with large 95% confidence interval.

FIG. 17: Evaluation of specific growth rate of nitrifying bacteria via accumulated NO_(x) concentration for wastewater and activated sludge from the WWTP utilizing extended aeration. The pH was held constant at 7 and reactors were aerated with air (control) or a mixture of air and pure CO₂ to operate at dissolved CO₂ of 7 mg/L (experimental).

FIG. 18: FISH Analysis Scale Representation. Scale bars equal 10 μm.

FIG. 19: Representative FISH images for nitrifying bacteria in MLE #1 including Nitrosomonas spp., Nitrosococcus mobilis (A); Nitrosospira spp. (B); Nitrobacter spp. (C); and most members of the phylum Nitrospirae (D).

FIG. 20: Representative FISH images for 4-Stage Bardenpho including Nitrosomonas spp., Nitrosococcus mobilis (A); Nitrosospira spp. (B); Nitrobacter spp. (C); and most members of the phylum Nitrospirae (D).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Biological ammonia removal in wastewater treatment plants is a slow process. This invention is the first to treat the dissolved CO₂ concentration and pH as important parameters in optimizing the specific growth rate of nitrifying bacteria. Five wastewater treatment plants (WWTP) representing the three major plant configurations, (1) extended aeration (EA), (2) Modified Ludzack-Ettinger (MLE), and (3) Bardenpho, were evaluated based upon their operating conditions and activated sludge properties. The specific growth rates of the nitrifying bacteria were calculated for field and optimal conditions for pH and dissolved CO₂ concentrations and suggest potential for improvement. Evaluation of nitrification in activated sludge at defined dissolved CO₂ concentrations and constant pH 7 verified these findings. Fluorescence in situ hybridizations (FISH) were used to determine the abundance of nitrifying bacteria populations in the activated sludge from each WWTP and lab-scale reactors. Changes in the community structure of the nitrifying bacteria demonstrate sensitivity to dissolved CO₂.

An important finding of this work is the high concentration of dissolved CO₂ in the aeration basins and other unit processes. Significant differences are evident and upon investigation are quite plausible. As an example, the aeration system on an MLE process uses three anoxic and four aerobic zones in a carousel arrangement to convert BOD and ammonia. A mixture of influent, RAS, and internal recycle from the aeration basin enter the anoxic basin, where denitrification generates additional dissolved CO₂ as a by-product. This treated wastewater with a high level of dissolved CO₂ then flows into the aeration basin where additional dissolved CO₂ is generated with minimal stripping. Evidence of the impact of anoxic treatment and minimal CO₂ stripping are observed in the MLE and Bardenpho systems. Plant influent also impacts the dissolved CO₂ concentration in the aeration basin and appears to be a function of the influent quality and collection system. Finally, the dissolved CO₂ concentration in the effluent is much higher than expected, when you consider that water in equilibrium with the atmosphere has a CO₂ concentration of 0.6 mg/L. It is unknown whether this elevated dissolved CO₂ concentration negatively impacts receiving water by providing a carbon source for the growth of algae and cyanobacteria.

Evaluation of the activated sludge from the WWTPs with Extended Aeration and MLE #1 showed differences in the specific growth rates of the nitrifying bacteria when the dissolved CO₂ concentration was optimized. The EA facility achieved a maximum growth rate at 7 mg/L CO₂ while the MLE #1 facility achieved a maximum growth rate at 12 mg/L, which are both near the optimal dissolved CO₂ concentration reported previously (Denecke and Liebig 2003). The community structure of the nitrifying bacteria in the activated sludge is expected to have a significant influence on the optimal dissolved CO₂ concentration. It should be noted that pH was held constant at 7 and optimization of the dissolved CO₂ concentration will increase the pH (FIG. 1).

The FISH results indicate differences in the community structure of the nitrifying bacteria amongst the WWTPs. Each facility appears to have its own established community of nitrifying bacteria. These results show that several AOB and NOB bacteria coexist in the same system, which is similar to a previous study (Siripong and Rittmann 2007). The four stage Bardenpho process, which operates near the ideal dissolved CO₂ concentration, shows a dominance of one AOB (Nitrosospira spp.) and NOB (phylum Nitrospirae). Due to its long SRT of nearly 26 days, the presence of other microbes is not unexpected. This suggests that as a process approaches the ideal dissolved CO₂ concentration for the growth of nitrifying bacteria, the community structure may becomes less diverse.

The differences in the observed presence of microbes among the WWTPs as seen in the FISH analysis (see Example V, Table 11) have one distinct possible cause. The community structure of the nitrifying bacteria may simply be different due to the influent variability. This is evident in observing the differences in the contribution of domestic wastewater in the influent between the plants. MLE #1 and MLE #2 have distinct variability in their AOB and NOB concentrations despite having essentially the same configuration and operational parameters. MLE #1 has a very low contribution of industrial wastewater, but is more diverse in the type of industrial wastewater it receives. MLE #2 has a large contribution of industrial wastewater, but consists mainly of wastewater from food processors as indicated by the high average BOD concentration.

FISH was used to investigate the nitrifying bacteria in lab-scale bioreactor experiments, which were conducted at dissolved CO₂ concentrations of 12 and 103 mg/L at a pH of 7.0. Compared to the seed material (MLE #1), the community structure of the nitrifying bacteria changed dramatically in unanticipated ways. Surprisingly, similar levels of Nitrosomonas spp. and Nitrospirae members were observed for both extreme dissolved CO₂ concentrations. However, levels of Nitrosospira spp. were much greater for the optimal dissolved CO₂ concentration and levels of Nitrobacter spp. were much greater for the suboptimal dissolved CO₂ concentration. In our attempts to provide optimal conditions for nitrification for the MLE #1 sludge, we were unable to produce a community structure of the nitrifying bacteria that was similar to the 4-stage Bardenpho. There may be several explanations for this failure. First, failure may be attributed to vastly different nitrifying bacteria in both samples, which would make it impossible to achieve this dominance of AOB and NOB populations present in the 4-stage Bardenpho. Second, it may be due to a lack of a wasting operation, which would remove slow-growing nitrifying bacteria. Third, we may be underestimating the difference in the effect of the influent wastewater properties. Fourth, we may be experiencing a pH effect, since the ideal dissolved CO₂ concentration increases the pH of the activated sludge to 7.56, which is more than half a pH unit above the lab-scale bioreactor experiment.

Methods to Implement in Full-Scale

Several methods exist to transform these findings into a technology for full-scale implementation. In most cases, the dissolved CO₂ concentration of the wastewater influent will need to be adjusted prior to pH adjustment and subsequent treatment in the aeration basin. The dissolved CO₂ concentration of the wastewater can be achieved by stripping the dissolved CO₂ from the influent or primary effluent. The pH can be adjusted through the addition of a strong base to all or a portion of the wastewater prior to treatment in the aeration basin. If the dissolved CO₂ concentration is too low in the aeration basin due to excessive stripping or transformation of dissolved CO₂ to the bicarbonate ion from base addition, the CO₂-enriched air stripped from the wastewater can be used as a source of CO₂ in the aeration system utilized in the aeration basin. Probes that measure the dissolved CO₂ concentration and pH can be positioned throughout the aeration basin to ensure optimal conditions for nitrification.

It is possible that higher rates of aeration in the aeration basin could reduce the soluble CO₂. Instead, a more cost effective means is the use of a CO₂ stripping stage to optimize the soluble CO₂ concentration for nitrification. Examples of such CO₂ stripping stages are air stripping towers and trickling filters.

A typical air stripping tower is a column equipped with a blower at the bottom of the column. The air stripping tower is vented to allow air and contaminants to vent to the outside. The blower blows air upward and the air removes the contaminants, here CO₂, from the water column. The upward air flow carries the CO₂ out the venting system. The air stripping tower can be filled with packing media as in a packed column. Therefore, the air stripping tower removes contaminants from water by cascading the water over a packing material designed to uniformly disperse the water throughout the tower while providing an upward flow of air which is also designed to uniformly disperse the water throughout the tower as well as provide a supply of air into which the contaminants may dissipate.

The conventional trickling filter utilizes a film of biomass fixed on a media to remove and aerobically convert organic matter to carbon dioxide, water and additional biomass and to oxidize ammonia to nitrates. The fixed media generally consists of rock, plastic or wood. Wastewater is distributed over the biomass fixed to media through an overhead rotary distributor having generally two to four nozzled arms or spreaders. This insures a relatively even distribution of wastewater over the fixed biomass and thereby produces a relatively constant loading throughout the filter area.

These technologies have been developed by the chemical and wastewater treatment industries. Traditionally, the air stripping tower has been utilized for high mass transfer rates of volatile organic compounds, while the trickling filter has been used as a fixed-film biological process for polishing secondary effluent. However, these technologies have not been employed for removal of soluble CO₂ for the express purpose of improving nitrification.

For MLE facilities (examples of which are shown in FIGS. 2 and 3), either technology could be used to process the internal recycle flow or the wastewater entering the aeration basin. The internal recycle flow rate is three times the influent wastewater flow rate and is used to provide a nitrate rich wastewater to the anoxic basin for denitrification. Either technology would reduce the soluble CO₂ levels, but would increase the dissolved oxygen content, which may inhibit denitrification in the anoxic tank and reduce performance.

When an increasing fraction of the internal recycle flow rate is treated by either technology, the soluble CO₂ concentration approaches the optimal CO₂ concentration for nitrification, as shown in FIG. 4. It is estimated that treatment of more than 50% of the internal recycle flow rate will yield no additional benefit with respect to the rate of nitrification.

If the air stripping technology is used for reducing and optimizing the soluble CO₂ concentration in the aeration basin, then the nitrification rate will allow for a 67% reduction in the hydraulic retention time in the aeration basin (FIG. 5) and tripling of the influent wastewater flow rate. In this configuration, solids removal of the internal recycle wastewater is assumed to be not necessary for CO₂ stripping. Additional capacity for anoxic treatment, secondary clarifier, and internal recycle pumps may be required (FIG. 6). Additional anoxic treatment capacity is possible by reducing the aeration rate in Aeration Basin #1. Enhanced aeration would be required in Aeration Basin #2 to provide adequate dissolved oxygen for carbonaceous BOD removal.

In another approach to optimizing the soluble CO₂ concentration, a separate nitrification treatment train, the existing aeration basins and secondary clarifiers are split into two serial activated sludge systems (FIG. 7). The first activated sludge system is operated for removal of cBOD, while the second activated sludge system is operated for nitrification. The cBOD treatment train requires a short HRT, which would be ideal for the retrofit of POTW with multiple aeration basins being operated in parallel. For example, some facilities operate six parallel aeration basins and three secondary clarifiers. In this approach, the CO₂ stripping technology could be used to treat the effluent from the secondary clarifier of the cBOD removal system.

In this configuration, solids removal of the internal recycle flow is necessary for CO₂ stripping. Enhanced aeration is required in the first train to provide adequate dissolved oxygen for cBOD removal.

In another embodiment, CO₂ stripping can occur between aeration basins for cBOD removal and nitrification. In this configuration, the CO₂ Stripping Technology is placed after cBOD removal and before the beginning of the Nitrification step (FIG. 8). This configuration improves soluble CO₂ concentrations for nitrification. In addition, the high dissolved oxygen concentration produced by the CO₂ stripping technology is advantageous for the nitrification step. This is a marked improvement compared to treating the internal recycle flow as shown in FIGS. 5 and 6. In this configuration, solids removal of the wastewater is assumed to be not necessary for CO₂ stripping. Enhanced aeration would be required in Aeration Basin #2 (bright green) to provide adequate dissolved oxygen for carbonaceous BOD removal.

Alternative Operational Conditions for SHARON Reactor

The Single reactor High activity Ammonia Removal Over Nitire (SHARON) process is based on the large difference in the specific growth rates of the AOB and NOB at elevated temperature (35° C.) and neutral pH (van Dongen, Jetten et al. 2001). The SHARON reactor is operated as a completely stirred tank reactor (CSTR) with an elevated temperature and low solids retention time. When operated in this manner, the NOB will eventually be washed out. The AOB will convert the ammonium to nitrite, which can be fed to downstream reactor, such as the ANAMMOX reactor. With the ANAMMOX reactor, a blend of equal parts ammonium and nitrite is converted under anoxic conditions to nitrogen gas. In another other operating mode, the SHARON reactor is operated as a sequencing batch reactor, where intermittent cycles of full aeration (nitrification to nitrite) and anoxic conditions with methanol addition (denitrification of nitrite to nitrogen gas) can completely remove the ammonium. Because of the elevated temperature requirement, the SHARON reactor is generally restricted to use with anaerobic digester supernatant which has a very high ammonium concentration and elevated temperature.

As shown in Table 1, an alternative to temperature has been identified as an alternative operating condition for the SHARON reactor. By operating at a dissolved CO₂ concentration of 14 mg/L and pH 8.2, the AOB grow faster (μ_(max,obs)=1.18 d⁻¹; maximum doubling time of 0.59 days) than the NOB (μ_(max,obs)=0.58 d⁻¹; maximum doubling time of 1.19 days). It is unknown whether these conditions may further improve the current operational strategy for the SHARON reactor. In other words, our results were obtained at room temperature (˜23° C.) and it is unclear whether the AOB maintains a significant advantage in specific growth rate compared to the NOB at elevated temperature (35° C.). However, the μ_(max,obs) of 1.18 d⁻¹ for the AOB with optimal pH and dissolved CO₂ is nearly 350% greater than the μ_(max,obs) calculated for the AOB in a SHARON reactor (0.26 d⁻¹) using their growth parameters for the optimal pH (7.23) and identical temperature (23° C.) (Van Hulle, Volcke et al. 2007). In addition, our low temperature μ_(max,obs) for the AOB is greater than the optimal the μ_(max,obs) for the AOB in the SHARON reactor (0.64 d⁻¹) operated at 35° C. and pH 6.8.

TABLE 1 Dissolved CO₂ concentration and pH for maximum μ_(max,obs) of ammonium oxidizing bacteria for alternative growth conditions for SHARON Reactor. optimal pH 8.20 optimal CO₂ 14.1 AOB High pH μ max, obs 1.18 pH term 0.56 CO₂ term 0.47 AOB Low pH μ max, obs 0.10 pH term 0.09 CO₂ term 0.50 NOB High pH μ max, obs 0.58 pH term 0.59 CO₂ term 0.25 NOB Low μ max, obs 0.09 pH term 0.09 CO₂ term 0.47

These results suggest that SHARON reactor operation may be possible with municipal wastewater, which is treated at 10-30° C. By optimizing the dissolved CO₂ concentration and pH of the municipal wastewater and reducing the solids retention time (SRT), the AOB should dominate in the aeration basin. If half of the municipal wastewater is treated in this manner, then the SHARON reactor treated municipal wastewater (nitrite rich) and untreated municipal wastewater could be blended together and fed to an ANAMMOX reactor downstream. There is interest in extending the SHARON/ANAMMOX reactor system for lower temperature operation, but the slow growth rate of the AOB in the SHARON reactor has been identified as the impediment (Vazquez-Padin, Fernadez et al. 2009).

EXAMPLES Example 1 Field Evaluation of Nitrification in Three BNR Systems

Five wastewater treatment plants (WWTP) representing the three major biological nutrients removal (BNR) configurations, were evaluated in this study that include an Extended Aeration, two MLE, 4-stage Bardenpho, and 5-stage Bardenpho. Dissolved CO₂ and pH were measured in each unit operation where dissolved CO₂ would be present. Dissolved CO₂ measurements were collected with the OxyGuard CO₂ meter. All pH values in the field were measured with an OAKTON pH Tester 10. Field measurements were collected during June and July 2009. All pH values in the laboratory were measured with an OAKTON model 510 pH meter.

An analysis of the three major types of wastewater treatment plant (WWTP) configurations was evaluated based on the dissolved CO₂ and pH of the influent, unit processes, and effluent (Table 2) and influent properties and operating conditions (Table 3). Dissolved CO₂ concentration and pH were the parameters of primary interest. The dissolved CO₂ values are representative of the measurements for the different unit processes. The pH values were determined by obtaining surface samples, which may not be representative for the particular unit process. As an example, the anoxic zone for the MLE #1 facility provided results ranging from 26 to 58 mg/L of dissolved CO₂ in its basin, where the probe was inserted 8-10 feet below the surface. Although the pH was reported as 7.35, we expected a pH of 6.7-7. None of the WWTPs receive anaerobic sludge brought in from other sources. Although the influent for the 4-stage Bardenpho process typically has a pH of 7.5, we recorded a pH of 7.9.

TABLE 2 Dissolved CO₂ concentration and pH of influent, unit processes, and effluent of five wastewater treatment plants. Extended 4-Stage 5-Stage Aeration MLE #1 MLE #2 Bardenpho Bardenpho CO₂ CO₂ CO₂ CO₂ CO₂ mg/L pH mg/L pH mg/L pH mg/L pH mg/L pH Influent 31 6.5 17 7.4 12 7.4 6 7.9 20 7.1 1° Clarifier N/A N/A 9 7.6 29 6.9 N/A N/A N/A N/A Anaerobic N/A N/A N/A N/A N/A N/A N/A N/A 31 7.0 Anoxic I 24 6.7 26-58 7.35^(a) 23-24 7.2 11 7.3 20 7.1 Aeration I 13.5^(a) 6.8 34 6.9 15-24 7.3^(a) 12 7.3 16 7.1 Anoxic II N/A N/A N/A N/A N/A N/A N/A N/A 22 7.0 Aeration II N/A N/A N/A N/A N/A N/A N/A N/A 23 7.0 2° Clarifier 12 6.9 23 7.1 23 7.3 11 7.4 19 7.2 Filtration N/A N/A 16 7.3 N/A N/A 6 7.5 12 7.4 Effluent 9 7.0 16 7.3 12 7.4 6 7.7 10 6.9 ^(a)the average of several measurements N/A: unit processes are not part of the configuration or were not in use.

Large differences in the influent dissolved CO₂ concentrations were observed among the WWTP. The influent of the extended aeration plant had a high dissolved CO₂ level but receives its influent through a large collection system where anaerobic conditions are quite probable and lead to these high readings. The 4-stage Bardenpho process, which has a low dissolved CO₂ concentration, is located in a residential community with a limited collection system. Little time is afforded for the influent to reach anaerobic conditions.

The MLE #2 exhibited a lower influent dissolved CO₂ concentration than observed in the primary clarifier. This WWTP is fed by a large underground piping system which suggests that anaerobic conditions are possible. On the day of the plant visit, a thunderstorm was in-progress and had increased the influent rate by 30 percent during the last hour. A diluted CO₂ influent concentration was recorded, while the primary clarifier had probably not seen the full effect of this dilution. In addition, the primary clarifier is covered and sealed tank, which may promote anaerobic activity.

The influence of the WWTP configuration is readily seen in the dissolved CO₂ concentration of the aeration basins. The dissolved CO₂ concentration in the anoxic basin is influenced by the mixture of the influent, internal recycled wastewater, and RAS combined with generation of dissolved CO₂ by denitrification. The 5-stage Bardenpho system has the additional contribution of dissolved CO₂ from the anaerobic treatment basin. This treated wastewater enters the aeration basin with an elevated dissolved CO₂ concentration that ranges from 11 to 58 mg/L. In the aeration basin, dissolved CO₂ is produced through the metabolism of the carbonaceous BOD by the heterotrophic bacteria, but dissolved CO₂ is also removed by stripping due to the intensive aeration.

The dissolved CO₂ concentration and pH were measured in unit processes beyond the activated sludge system. All WWTP are discharging final effluent with elevated dissolved CO₂ concentrations when compared to the dissolved CO₂ concentration of water in equilibrium with the atmosphere (0.6 mg/L). The elevated level of dissolved CO₂ is not surprising since the terminal unit processes do not provide adequate stripping.

TABLE 3 Influent properties and activated sludge operating conditions for five wastewater treatment plants. Extended MLE MLE 4-Stage 5-Stage units Aeration #1 #2 Bardenpho Bardenpho Domestic 100% 95% 81% 100% 95% WW BOD mg/L 300 200 550 207 200 NH₄ ⁺—N mg/L 25 28 25 35 31 COD mg/L 587 N/A 1,250 N/A N/A MLSS mg/L 3,190 2,900 4,092 2,815 3,200 MLVSS mg/L 2,490 2,320 3,384 2,252 2,240 SRT days 17 12 9 25.9 15 Aeration mg/L 1-3 2-5 1.5-3 0.8-1.2 0.4 DO N/A: Not available.

MLE #2 has the lowest domestic wastewater percentage of all the plants evaluated. It services major food processing industries as indicated by its high influent BOD and COD, which requires an elevated solids concentration (MLSS) to ensure proper treatment.

The dissolved oxygen (DO) concentrations are markedly different among the WWTPs. The extended aeration and the MLE plants show expected DO levels typically encountered at wastewater facilities. The Bardenpho processes utilize reduced DO levels to achieve their BOD and ammonia conversions as higher DO concentrations interfere with conversion in their anoxic and anaerobic zones.

Example II pH vs. Dissolved CO₂

An activated sludge sample was collected from the aeration basin of each WWTP evaluated. Within one hour of collection, the sample was evaluated in the laboratory to determine the pH at varying dissolved CO₂ concentrations. The sample was placed in a one liter beaker in a sealed desiccant cabinet and air or an air/CO₂ mixture was introduced into the cabinet. An air pump inside the cabinet subsequently introduced the atmosphere into the beaker. The atmosphere was maintained for a minimum of 15 minutes at which time dissolved CO₂ and pH were measured.

The experiment was conducted to determine the maximum specific growth rate at combinations of three selected dissolved CO₂ concentrations (5, 10 and 15 mg/l) and four pH levels (6.5, 7.0, 7.5 and 8.0). Desiccant cabinets were fabricated that maintained the appropriate pCO₂ with four reactors placed inside the cabinets at the appropriate pH. All reactors were operated at room temperature (23° C.). Phosphate buffer was used to control pH with minor additions of base or acid for adjustment at the pH 8 and 6.5 conditions. Nitrite and nitrate concentration measurements were taken at 1.5 hour intervals, 18-20 hours per day for approximately 10 days. The plots of the nitrate (NO₃ ⁻—N) and NO_(x) (NO₂ ⁻—N+NO₃ ⁻—N) for each experimental reactor are provided in FIGS. 9A-11D.

From these plots, it is clear that there are some gross differences in the rate of growth of the AOB and NOB populations, especially at higher pH. Other plots suggest lower rate of growth for low dissolved CO₂ concentration and pH. These data were analyzed by the use of a non-linear regression software application, which estimated the maximum, observed specific growth rate (μ_(max,obs)) for the AOB and NOB populations (FIG. 12).

These results suggest a significant improvement in AOB μ_(max,obs) is possible by providing pH 8 and dissolved CO₂ between 10-15 mg/L. A large difference in the μ_(max,obs) was observed between the AOB and NOB for these conditions. The increase in the μ_(max,obs) at lower pH and dissolved CO₂ concentrations suggested the presence of two AOB and NOB populations. The AOB and NOB population were split into Low and High pH subpopulations and the growth parameters were determined (Table 4 and FIGS. 13-16).

TABLE 4 Estimated growth parameters μmax, CO₂ and pH) for two ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) populations in MLE #1 activated sludge. AOB High pH NOB High pH μ max 4.5 μ max 4.0 pH Term pH pH Term pH K1 1.58E−08 7.8 K1 3.16E−08 7.5 K2 2.51E−09 8.6 K2 3.16E−09 8.5 CO₂ term CO₂ term KCO₂ 8 KCO₂ 3 KI 25 KI 5 AOB Low pH NOB Low pH μ max 2.1 μ max 2.0 pH Term pH pH Term pH K1 6.31E−07 6.2 K1 6.31E−07 6.2 K2 6.31E−08 7.2 K2 6.31E−08 7.2 CO₂ term CO₂ term KCO₂ 10 KCO₂ 12 KI 50 KI 50

The growth parameters for both AOB and NOB populations were used to estimate the improvement in the μ_(max,obs) for the complete nitrification of ammonium for different wastewater treatment plants that optimize dissolved CO₂ concentration (5.5 mg/L) without optimization of the pH (Table 5). The corresponding pH was extrapolated for the optimized dissolved CO₂ concentration by using the data used for generating FIGS. 9A-D. For this analysis, the μ_(max,obs) of the nitrifier (AOB or NOB) that limits the overall nitrification reaction for the existing and optimized condition were compared directly. This analysis suggests that four of the seven systems may have greater rates of complete nitrification by optimizing the dissolved CO₂ concentration. The greatest improvement was predicted for the 4-stage BardenPho system. When the dissolved CO₂ concentration (5.5 mg/L) and pH (8.07) were optimized, the improvement in the μ_(max,obs) were predicted for all seven systems with a range of 59-122% (Table 6).

TABLE 5 Estimated improvement of μ_(max,obs) of nitrifying bacteria of six wastewater treatment plants with six unique BNR configurations for complete nitrification through optimal dissolved CO₂ concentration and corresponding pH. Modified High Purity O₂ Extended Ludzack-Ettinger BardenPho cBOD Aeration #1 #2 4-stage 5-stage Removal Nitrification Actual pH 6.8 6.9 7.3 7.3 7.1 6.8 6.8 Actual CO₂ 13.5 34 20 12 16 64 40 μ max, obs actual 0.63 0.63 0.49 0.50 0.56 0.52 0.62 μ max, obs LIMITING 0.56 0.58 0.43 0.41 0.50 0.49 0.58 AOB High pH 0.19 0.19 0.49 0.50 0.35 0.11 0.15 pH term 0.09 0.11 0.24 0.24 0.17 0.09 0.09 CO₂ term 0.47 0.39 0.45 0.47 0.47 0.27 0.36 AOB Low pH 0.63 0.63 0.47 0.43 0.56 0.52 0.62 pH term 0.61 0.59 0.43 0.43 0.52 0.61 0.61 CO₂ term 0.50 0.51 0.53 0.48 0.51 0.41 0.49 NOB High pH 0.17 0.10 0.29 0.41 0.26 0.05 0.07 pH term 0.17 0.20 0.38 0.38 0.28 0.17 0.17 CO₂ term 0.25 0.13 0.19 0.27 0.23 0.07 0.11 NOB Low pH 0.56 0.58 0.43 0.38 0.50 0.49 0.58 pH term 0.61 0.59 0.43 0.43 0.52 0.61 0.61 CO₂ term 0.46 0.49 0.50 0.45 0.48 0.41 0.48 corresponding PH 7.52 7.54 7.49 7.87 7.68 7.62 7.62 optimal CO₂ 5.5 5.5 5.5 5.5 5.5 5.5 5.5 μ max, obs optimal 0.73 0.75 0.71 0.91 0.83 0.80 0.80 μ max, obs LIMITING 0.56 0.58 0.54 0.83 0.69 0.64 0.64 AOB High pH 0.56 0.58 0.54 0.83 0.69 0.64 0.64 pH term 0.33 0.34 0.32 0.49 0.41 0.38 0.38 CO₂ term 0.37 0.37 0.37 0.37 0.37 0.37 0.37 AOB Low pH 0.23 0.22 0.24 0.13 0.18 0.20 0.20 pH term 0.32 0.31 0.33 0.18 0.25 0.27 0.27 CO₂ term 0.34 0.34 0.34 0.34 0.34 0.34 0.34 NOB High pH 0.73 0.75 0.71 0.91 0.83 0.80 0.80 pH term 0.49 0.49 0.47 0.60 0.55 0.53 0.53 CO₂ term 0.38 0.38 0.38 0.38 0.38 0.38 0.38 NOB Low pH 0.19 0.19 0.20 0.11 0.15 0.17 0.17 pH term 0.32 0.31 0.33 0.18 0.25 0.27 0.27 CO₂ term 0.30 0.30 0.30 0.30 0.30 0.30 0.30 % improvement 16% 20% 47% 83% 48% 53% 29% MAX % improvement 0% 0% 26% 100% 37% 31% 11% LIMITING

TABLE 6 Estimated improvement of μ_(max,obs) of nitrifying bacteria of six wastewater treatment plants with six unique BNR configurations for complete nitrification through optimal dissolved CO₂ concentration and pH. Modified High Purity O₂ Extended Ludzack-Ettinger BardenPho cBOD Aeration #1 #2 4-stage 5-stage Removal Nitrification pH 6.8 6.9 7.3 7.3 7.1 6.8 6.8 CO₂ 13.5 34 20 12 16 64 40 μ max, obs actual 0.63 0.63 0.49 0.50 0.56 0.52 0.62 μ max, obs Limiting 0.56 0.58 0.43 0.41 0.50 0.49 0.58 AOB High pH 0.19 0.19 0.49 0.50 0.35 0.11 0.15 pH term 0.09 0.11 0.24 0.24 0.17 0.09 0.09 CO₂ term 0.47 0.39 0.45 0.47 0.47 0.27 0.36 AOB Low pH 0.63 0.63 0.47 0.43 0.56 0.52 0.62 pH term 0.61 0.59 0.43 0.43 0.52 0.61 0.61 CO₂ term 0.50 0.51 0.53 0.48 0.51 0.41 0.49 NOB High pH 0.17 0.10 0.29 0.41 0.26 0.05 0.07 pH term 0.17 0.20 0.38 0.38 0.28 0.17 0.17 CO₂ term 0.25 0.13 0.19 0.27 0.23 0.07 0.11 NOB Low pH 0.56 0.58 0.43 0.38 0.50 0.49 0.58 pH term 0.61 0.59 0.43 0.43 0.52 0.61 0.61 CO₂ term 0.46 0.49 0.50 0.45 0.48 0.41 0.48 optimal pH 8.07 8.07 8.07 8.07 8.07 8.07 8.07 optimal CO₂ 5.5 5.5 5.5 5.5 5.5 5.5 5.5 μ max, obs Max 0.92 0.92 0.92 0.92 0.92 0.92 0.92 μ max, obs Limiting 0.92 0.92 0.92 0.92 0.92 0.92 0.92 AOB High pH 0.92 0.92 0.92 0.92 0.92 0.92 0.92 pH term 0.55 0.55 0.55 0.55 0.55 0.55 0.55 CO₂ term 0.37 0.37 0.37 0.37 0.37 0.37 0.37 AOB Low pH 0.09 0.09 0.09 0.09 0.09 0.09 0.09 pH term 0.12 0.12 0.12 0.12 0.12 0.12 0.12 CO₂ term 0.34 0.34 0.34 0.34 0.34 0.34 0.34 NOB High pH 0.92 0.92 0.92 0.92 0.92 0.92 0.92 pH term 0.61 0.61 0.61 0.61 0.61 0.61 0.61 CO₂ term 0.38 0.38 0.38 0.38 0.38 0.38 0.38 NOB Low pH 0.07 0.07 0.07 0.07 0.07 0.07 0.07 pH term 0.12 0.12 0.12 0.12 0.12 0.12 0.12 CO₂ term 0.30 0.30 0.30 0.30 0.30 0.30 0.30 % improvement 46% 47% 90% 85% 64% 76% 48% MAX % improvement 63% 59% 115% 122% 83% 87% 59% LIMITING

Example III Specific Growth Rate Measurement in Lab-Scale Bioreactors

The experiments were conducted based upon previously published guidelines (Melcer et al. 2003). Two 3 liter beakers were used for the reactors. The control reactor utilized air, while the experimental reactor was aerated with a mixture of air and pure CO₂ to produce dissolved CO₂ concentrations of 12 and 103 mg/L. The pH was maintained between 7.0±0.05 through the addition of a phosphate buffer. Each reactor received identical phosphate buffer additions.

Both reactors were fed influent from the MLE #1 WWTP. A series of preliminary experiments were conducted to establish appropriate operating conditions. Based on these results, 60 mg/L of NH₄ ⁺—N was added to the influent wastewater which contained, on average, 25 mg/L of NH₄ ⁺—N. The dissolved oxygen was held constant at 8.3 mg/L as O₂, which ensured that oxygen was not limiting. Each reactor had an initial addition of 0.5 grams of sodium bicarbonate with 0.5 gram additions during the reaction sequence based on NH₄ ⁺—N conversion.

Each reactor was inoculated with activated sludge that was collected from the aeration basin of the MLE #1's activated sludge system on the same day that the experiment was initiated. A MLVSS target value of 35 mg/L was specified in these experiments. Throughout the experiment, NH₄ ⁺, NO₂ ⁻, NO₃ ⁻, pH, and dissolved oxygen (DO) were routinely measured. A non-linear regression model was used to regresses the NO_(x) ⁻ concentration levels (NO₂ ⁻+NO₃ ⁻) versus time and estimate specific growth rate, μ, of the nitrifying bacteria by using a software package (Oakdale Engineering).

An initial study of the effect of dissolved CO₂ concentration on the specific growth rate of nitrifying bacteria was conducted using activated sludge from the extended aeration facility. The results of an analysis with pH 7.0 and CO₂ concentration at 7 mg/L versus air are provided (FIG. 17). The selection of the 7 mg/L dissolved CO₂ (0.4%) concentration was based on previous research (Denecke and Liebig 2003).

Both reactors display a buildup of NO_(x) ⁻ concentration (NO₂ ⁻ and NO₃ ⁻) over a 10 day period. However, it is evident that the rate of NO_(x) ⁻ concentration buildup is significantly higher in the experimental reactor. The specific growth rate of the nitrifying bacteria was estimated by fitting the non-linear response. The specific growth rate, μ for both conditions and the associated 95% confidence interval are provided in Table 7. The regression analysis was conducted to NO_(x) values of approximately 20 mg/L. Inhibition effects were observed at values greater than this concentration (data not shown).

TABLE 7 Estimated specific growth rate of nitrifying bacteria and 95% confidence interval of the activated sludge from the WWTP with extended aeration for two defined dissolved CO₂ concentrations. Dissolved CO₂ μ Lower Upper Reactor (mg/L) (days⁻¹) Limit Limit Control 0.6 0.578 0.479 0.677 Experimental 7 1.011 0.802 1.219

Further research was conducted using activated sludge from the WWTP with MLE #1. The sludge was evaluated at varying levels of pCO₂ from 7 to 17 mg/L at a constant pH of 7. An optimum specific growth rate of 0.84 days⁻¹ was achieved at a dissolved CO₂ of 12 mg/L.

Example IV Estimation of Specific Growth Rate of Nitrifying Bacteria

Growth rate optimization was based on Monod kinetics. The Haldane equation was used to determine the effect of the dissolved CO₂ concentration on the specific growth rate. The pH sensitivity of the specific growth rate was calculated by using an optimal pH of 8 as reported optimum values range from 7.5 to 8.5 (Metcalf & Eddy 2003). Specific growth rate optimization was based on results previously reported (Denecke and Liebig 2003). The parameters and coefficients are provided in Table 8.

TABLE 8 Constants used to calculate the optimal specific growth rate for nitrifying bacteria. Constant Value K_(CO2), mg/L 0.5 Ki, mg/L 42 K₁ for pH 1.58E-07 K₂ for pH 6.31E-10 μ_(max) 0.75 b 0.1 pH Term Max 0.88 CO₂ Term Max 0.82

As disclosed herein, the relationship between the maximum specific growth rate of the nitrifying bacteria and corresponding dissolved CO₂ can be described by the Andrew's equation. The Andrew's equation is similar to the traditional Monod equation, which describes the specific growth rate as a function of the true maximum specific growth rate and a single rate limiting substrate. However, the Andrew's equation shown below includes an additional inhibition term that reduces the specific growth rate for high concentrations of the substrate.

$\mu = {\mu_{\max}\frac{\left\lbrack {CO}_{2} \right\rbrack}{\left( {\left\lbrack {CO}_{2} \right\rbrack + K_{CO2} + \frac{\left\lbrack {CO}_{2} \right\rbrack^{2}}{K\; 1}} \right)}}$

In addition to the CO₂ term described, the specific growth rate is also adjusted for the pH by the following term, where K1 and K2 are constants specific for a bacterial species.

$\underset{term}{{pH}\mspace{14mu}{correction}} = \left( \frac{1}{1 + {{\left\lbrack H^{+} \right\rbrack/K}\; 1} + {K\;{2/\left\lbrack H^{+} \right\rbrack}}} \right)$

An overall expression for μ_(max,obs) includes μ_(max) and terms that correct for dissolved CO₂, pH, ammonium, dissolved oxygen (DO), and temperature The pH and dissolved CO₂ terms are normalized to generate values between 0-1 by dividing the equations above by the maximum term value. In this way, all correction terms have values between 0-1 and can be multiplied together to adjust μ_(max,obs.) μ_(max,obs)=μ_(max)(CO₂ term)(pH term)(N term)(DO term)(Temp term)

A simpler expression is provided, which condenses μ_(max) and terms that correct for dissolved CO₂ and pH. μ_(max,obs)=μ_(max,CO2,pH))(N term)(DO term)(Temp term)

The formula to determine the field and optimum specific growth rate of the nitrifying bacteria is provided:

${\mu obs} = {{{\mu max} \times \frac{\frac{\left\lbrack {CO}_{2} \right\rbrack}{\left\lbrack {CO}_{2} \right\rbrack + {Ks} + \frac{\left\lbrack {CO}_{2} \right\rbrack^{2}}{Ki}}}{{CO}_{2}{Term}\mspace{14mu}{Max}} \times \frac{\frac{1}{\left( {1 + \frac{\left\lbrack H^{+} \right\rbrack}{K\; 1} + \frac{K\; 2}{\left\lbrack H^{+} \right\rbrack}} \right)}}{{pH}\mspace{14mu}{Term}\mspace{14mu}{Max}}} - b}$

The CO₂ term max is the value obtained at a dissolved CO₂ value of 5 mg/L. The pH term max is the value obtained at a pH of 8. These values are used to normalize the formula by using the maximum specific growth rate for ideal dissolved CO₂ concentration and pH. Denecke reported that a 5 mg/L dissolved CO₂ concentration is equivalent to 0.4% CO₂. When calculated using Henry's constant, 0.4% equates to 6.89 mg/L. For purposes of this study, 5 mg/L was used as the optimum CO₂ concentration. Field pH measurements used in this study were calculated from activated sludge evaluated at varying levels of CO₂ concentrations in the laboratory. Although actual field measurements are reported later in this paper, there was concern as to how well they represented actual pH values at the specified dissolved CO₂ concentrations.

A sample of activated sludge from the aeration basin of each process was obtained and evaluated at different dissolved CO₂ concentrations (FIG. 1). The numbers in the figure represent the dissolved CO₂ concentrations in the aeration basin for the WWTP.

Results show a general downward trend (lower pH) with increasing levels of CO₂. Although different configuration types appear to segregate, this difference maybe more related to their MLVSS concentrations. When the dissolved CO₂ concentration is reduced for activated sludge with elevated levels, the pH also increases. For example, the reduction of the dissolved CO₂ concentration to 15 mg/L would produce pH of 7.1-7.5.

Each WWTP was further evaluated to determine the potential for increasing the specific growth rate of the nitrifying bacteria by optimizing the dissolved CO₂ concentration and allowing for pH adjustment (FIG. 1). Our results, shown in Table 9, suggest that improvements are possible for each WWTP evaluated in this study with the MLE facilities offering the greatest potential. The Bardenpho processes offer less potential for improvement due to the low dissolved CO₂ concentrations and higher operating pH values, which are near the optimum values.

TABLE 9 Optimum specific growth rate of nitrifying bacteria for optimal dissolved CO₂ concentration of 5 mg/L and corresponding pH. Extended MLE MLE 4-Stage 5-Stage Properties Aeration #1 #2 Bardenpho Bardenpho CO₂, field 14 34 20 12 16 pH, field 7.17 6.92 7.01 7.57 7.26 pH, corresponding 7.54 7.56 7.51 7.89 7.7 to optimal CO₂ μ, observed 0.4238 0.22 0.3226 0.5501 0.4368 μ, optimum 0.6016 0.6058 0.595 0.6473 0.6297 μ, Improvement 42% 175% 84% 18% 44%

Example V Evaluation of Nitrifying Bacteria Abundance by Fluorescence In Situ Hybridization

Four fluorescently-labeled oligonucleotide hybridization probes, that target two ammonia oxidizing bacteria (AOB) and nitrifying oxidizing bacteria (NOB) groups were used in this study (Table 10) were synthesized and conjugated with the cyanine dye, Cy3, before purification with oligonucleotide probe purification cartridges. Fluorescently labeled probes were diluted to 50 ng/μL with RNase-free water and stored at −20° C. in the dark. Samples (1 mL) were collected from the aeration basin from each WWTP and fixed with 1 mL of 4% PFA for 12-24 hours. The samples were centrifuged and supernatant decanted, and resuspended in 2 mL of ethanol PBS (EtOH-PBS). The samples were stored at −20° C. until further analysis. Fixed samples were applied to a sample well on a 10 well Heavy Teflon Coated microscope slide (Cel-Line Associates, New Field, N.J.) and air-dried. After dehydration with an increasing ethanol series (50, 80, 95% [vol/vol]ethanol, 1 min each), each sample well was covered with a mixture of 18 μL of hybridization buffer (20% [vol/vol] formamide, 0.9 M NaCl, 100 mM Tris HCl [pH 7.0], 0.1% SDS) (DelosReyes et al. 1997) and 2 μL of the stock fluorescently labeled oligonucleotide probe. The hybridizations were conducted in a moisture chamber containing excess hybridization buffer (to prevent dehydration of buffer on sample wells) for 1.5 h, in the dark, at 46° C. The slides were washed for 30 min at 48° C. with 50 mL of pre-warmed washing buffer solution (215 mM NaCl, 20 mM Tris HCl [pH 7.0], 0.1% SDS, and 5 mM EDTA) (DelosReyes et al. 1997). Fixed, hybridized cells were mounted with Type FF immersion oil (Cargille, Cedar Grove, N.J.) and a cover slip. Cells were stained with 4′, 6-diamidino-2-phenylindole (DAPI) at a concentration of 1 μg/mL for 1 minute and rinsed with DI water.

TABLE 10 FISH probe information. Probe Targeted bacteria Reference AOB NSM156 Nitrosomonas spp., (Mobarry et al. 1996) Nitrosococcus mobilis Nsv433 Nitrosospira spp. (Mobarry et al. 1996) NOB NIT3 Nitrobacter spp. (Wagner et al. 1996) Ntspa0712 most members of the (Daims et al. 2001) phylum Nitrospirae

Whole cell fluorescence was visualized with an upright epiflourescence microscope (Leitz DiaPlan, Heerbrugg, Switzerland), and digital images were captured using a Spot-FLEX charge coupled device (CCD) camera (Diagnostic Instruments, Inc., Sterling Heights, Mich.). Images were collected using a 100× oil objective and constant exposure time of 1.2 sec and gain of 2. For each FISH probe, ten images were collected for each sample and analyzed based on the relative abundance of cy3 fluorescent cells. Direct measurement of abundance was difficult due to the background fluorescence of the samples, thus a simple scale (FIG. 18) was used to estimate the abundance. The value of each set of images was totaled and averaged.

Representative FISH images for the samples collected from the MLE #1 and the 4-stage Bardenpho are provided in FIGS. 19 and 20. Individual cells and small clusters of cells are present in the flocs for each of the major ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB). Frequent background fluorescence made enumeration difficult, which required a more qualitative approach that utilized a relative abundance scale (FIG. 18).

Analysis of the digital FISH images using the relative abundance scale is provided in Table 11. These values show a strong presence of each major AOB and NOB in each WWTP. The extended aeration system appears to have the lowest abundance of AOB and NOB compared to the other WWTPs, although it appears to have a similar community structure to the 5-stage Bardenpho. The two MLE samples have similar NOB community structure; however the AOB appear to be inverted.

Attempts to alter the specific growth rate of the nitrifying bacteria by operation at extreme dissolved CO₂ concentrations of 12 and 103 mg/L produced interesting results. For optimal dissolved CO₂ concentration (12 mg/L), the AOB populations appear to be even, while the Nitrosomonas spp. appears to dominate the Nitrosospira spp. amongst the NOB. For the extreme suboptimal dissolved CO₂ concentration (103 mg/L), the Nitrosomonas spp. dominate the Nitrosospira spp. for the AOB and the NOB populations are higher but more even compared to the field sample. When compared to each other, the abundance of the Nitrosomonas spp. and Nitrospirae spp. appear to be similar, while Nitrosospira spp. are much higher for the reactor operating under optimal CO₂ concentration and the Nitrobacter spp. are much higher for the reactor operating under suboptimal CO₂ concentration.

A careful review of the dissolved CO₂ and pH values suggest that the 4-stage Bardenpho system should be operating at near optimal conditions for nitrification. In this system, the dominant AOB appears to be the Nitrosospira spp. and the dominant NOB appears to be the Nitrospirae phylum. In contrast, the 5-stage Bardenpho system has a higher abundance of Nitrosomonas spp., but the Nitrosospira spp. are still dominant amongst the NOB. The members of the phylum Nitrospirae are much lower relative to the 4-stage Bardenpho, while the Nitrobacter spp. are similar.

TABLE 11 FISH Analysis of five WWTP and lab-scale reactors operated at extreme dissolved CO₂ concentrations. MLE #1 12 mg/L 103 mg/L MLE 4-Stage 5-Stage EA Field CO₂* CO₂* #2 BP BP AOB NSM156 2.40 3.30 4.90 5.00 5.60 1.00 3.50 Nitrosomonas spp., Nitrosococcus mobilis Nsv433 4.50 5.60 4.80 3.30 4.50 5.00 5.40 Nitrosospira spp. NOB NIT3 2.73 7.20 4.00 6.20 6.73 2.80 2.44 Nitrobacter spp. Ntspa717 1.90 5.80 6.00 6.80 5.50 5.00 1.70 most members of the phylum Nitrospirae *pH 7

It will be seen that the advantages set forth above, and those made apparent from the foregoing description, are efficiently attained and since certain changes may be made in the above construction without departing from the scope of the invention, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween. Now that the invention has been described, 

What is claimed is:
 1. A wastewater treatment system comprising: at least one anoxic basin having an input and an output; at least one aerobic basin in fluid communication with the output of the at least one anoxic basin; wherein the at least one aerobic basin has an input and an output; a recycle line having an input in fluid communication with the aerobic basin or in fluid communication with the output from the at least one aerobic basin, and an output in fluid communication with the anoxic basin input; a CO₂ stripping stage disposed on the recycle line; and a clarifier in fluid communication with the output from the at least one aerobic basin and disposed downstream from the recycle line.
 2. The system of claim 1, further comprising a plurality of secondary clarifiers in fluid communication with the output from the at least one aerobic basin.
 3. The system of claim 2, wherein the output sludge from the plurality of secondary clarifiers is in fluid communication with the input of the at least one anoxic basin.
 4. The system of claim 1, further comprising an internal recycle pump in communication with the output from the at least one aerobic basin.
 5. The system of claim 1, further comprising a first secondary clarifier disposed between the aerobic basin and the CO₂ stripping stage.
 6. The system of claim 1, further comprising a nitrification stage disposed after the CO₂ stripping stage.
 7. The system of claim 6, further comprising a second secondary clarifier disposed after the nitrification stage.
 8. The system of claim 1, wherein the CO₂ stripping stage is selected from the group consisting of an air-stripping tower and a trickling filter.
 9. The system of claim 8, wherein the air-stripping tower further comprises: a column; a blower disposed at the bottom of the column; and a plurality of vents disposed to allow air and contaminants to vent to the outside.
 10. The system of claim 8, wherein the trickling filter further comprises: a film of biomass fixed on a media, wherein the media is rock, plastic or wood; and a rotary distributor disposed to distribute wastewater over the film of biomass.
 11. The system of claim 10, wherein the rotary distributor is an overhead distributor having two to four nozzled arms or spreaders.
 12. A wastewater treatment system comprising: at least one anoxic basin having an input and an output; a first plurality of aerobic basins in fluid communication with the output of the at least one anoxic basin; wherein the first plurality of aerobic basins has an output; a transfer line having an input and output, wherein the input of the transfer line is in fluid communication with the output from the first plurality of aerobic basins; a CO₂ stripping stage disposed on the transfer line; a second plurality of aerobic basins having an input and an output, wherein the input from the second plurality of aerobic basins is in fluid communication with the output of the transfer line; and a recycle line having an input in fluid communication with the output from the second plurality of aerobic basins and an output in fluid communication with the anoxic basin input.
 13. The system of claim 12, further comprising a plurality of secondary clarifiers in fluid communication with the output from the second plurality of aerobic basins.
 14. The system of claim 12, further comprising an internal recycle pump in communication with the output from the second plurality of aerobic basins.
 15. The system of claim 12, wherein the CO₂ stripping stage is selected from the group consisting of an air-stripping tower and a trickling filter.
 16. The system of claim 15, wherein the air-stripping tower further comprises: a column; a blower disposed at the bottom of the column; and a plurality of vents disposed to allow air and contaminants to vent to the outside.
 17. The system of claim 15, wherein the trickling filter further comprises: a film of biomass fixed on a media, wherein the media is rock, plastic or wood; and a rotary distributor disposed to distribute wastewater over the film of biomass. 